Fluorescence microscope arrangement

ABSTRACT

The invention relates to a microscope arrangement and to a method with which the spatial distribution of a magnetically and/or electrically sensitive fluorescent marker ( 21 ) in a sample ( 20 ) can be determined. Fluorescence radiation (VF) is excited by primary radiation (VE) in the sample ( 20 ) and imaged by a microscope. At the same time, within the sample ( 20 ) a spatially inhomogeneous magnetic and/or electric field ( 33 ) is generated, which has, for example, a small focal region ( 22 ) of minimum field strength. The emission of fluorescence radiation is locally modified in the focal region ( 22 ), which can be observed in the measured intensity distribution (IFM). In this way, the distribution of the fluorescence marker ( 21 ) even in regions ( 22 ) having a size below the optical resolution of the microscope ( 10 ) can be reconstructed.

The invention relates to a microscope arrangement having a fluorescencemicroscope for imaging the distribution of a fluorescent marker insample. The invention relates furthermore to a method for determiningthe spatial distribution of a fluorescent marker in a sample.

In fluorescence microscopy, the distribution of a fluorescent marker ina sample is observed with a microscope designed for that purpose. Afluorescent marker is a chemical substance, which, after being excitedby suitable primary radiation, emits fluorescent light of acharacteristic spectral range, which is why the term “fluorescent dye”is often used for it. By coupling a fluorescent marker to othermolecules, such as medicaments or proteins, information can be obtained,for example, about metabolic processes in biological systems. But theresolution that can be achieved with current fluorescence microscopes islimited to a range of about 100 nm to 30 nm, so that the study ofrelatively small structures or of processes on a molecular level is notpossible.

Against this background, it was an object of the present invention toprovide means for improving the resolution in fluorescence microscopy inclear and turbid media.

That object is achieved by a microscope arrangement having the featuresof claim 1 and by a method having the features of claim 5. Advantageousembodiments are contained in the subsidiary claims.

The microscope arrangement according to the invention is used forimaging a sample (for example, a biological sample) that contains afluorescent marker. Here, the fluorescent marker shall be one that ismagnetically and/or electrically sensitive, that is, with which thefluorescence behavior (fluorescence intensity, spectral shifts offluorescence, polarization, variation in time of the intensity etc.) ofthe marker is influenced by an external magnetic or electric field.

The microscope arrangement contains the following components:

A fluorescence microscope for exciting and imaging fluorescenceradiation from the sample. Suitable microscopes for this purpose areknown from the field of fluorescence microscopy.

A field generator for generating a spatially inhomogeneous magneticand/or a spatially inhomogeneous electric field in the sample, whereinthe inhomogeneity of the field must be present at least in a localregion.

Using the described microscope arrangement, it is possible to extractmore information from the study of a sample than with conventionalfluorescence microscopy. This is attributable to the fact that it ispossible to generate in the sample a spatially inhomogeneous field that,in accordance with requirements, influences the emission behavior of thefluorescent marker to be observed. The field therefore gives the userthe opportunity to vary the local conditions within the samplespecifically and consequently to influence the fluorescence. Inparticular, in this way the resolution of the microscope arrangement inrespect of the fluorescence radiation can be improved, specificapplications of the arrangement being described in more detail below.Furthermore, using the microscope arrangement it is possible also tocarry out analyses in turbid media with improved resolution.

According to a first, preferred embodiment, the microscope arrangementis designed to modify in a defined manner the inhomogeneous magneticand/or electric field within the sample, for example, to shift itsposition and/or to change its distribution. By observing how a givensample volume responds to the change in the field, important informationcan be obtained about the fluorescent marker contained therein. If, forexample, the field has a small focal region with special conditions(e.g. a minimum of field strength), then, with this, selectively atdifferent points within the sample the presence of the fluorescentmarker can be analyzed.

There are various possibilities for constructing a field generatorhaving the desired properties. In this regard to some extent one canhave recourse to solutions that are known for other applications, suchas, for example, the imaging of magnetic particles (cf. DE 101 51 778A1, which by reference is incorporated in its entirety into the presentapplication). According to a preferred embodiment, the field generatorcomprises a first pole body of a first polarity (in the case of magneticfields, for example “North”, in the case of electric fields, forexample, “negative”), which on at least two opposite sides is adjacentto second pole bodies of the other polarity (“South” respectively“positive”). The first pole body preferably has a tip. As will beexplained in detail within the scope of the description of the drawings,in the case of such a configuration there is generally a point-formregion in the vicinity of the field generator at which the fieldstrength assumes a minimum. This is then suitable as a focal region whenobserving a sample.

The changes in fluorescence behavior in the sample caused by themagnetic or electric field can in the simplest case be observed by theuser of the microscope arrangement purely by eye. Preferably, however,an advanced image processing of the image recorded with the fluorescencemicroscope takes place by means of a data processing device. In thisregard, the data processing device is designed to reconstruct thedistribution of the fluorescent marker in the sample from the knownstrength distribution of the (spatially and optionally also temporally)inhomogeneous field during one or more recordings and from the measuredfluorescence radiation. If, for example, the field has a focal region ofminimal field strength, the data processing device is able to take intoaccount the fact that the fluorescence in this region is correspondinglychanged (increased or reduced).

The invention furthermore relates to a method for determining thespatial distribution of a magnetically and/or electrically sensitivefluorescent marker in a sample, which method comprises the followingsteps:

Generation of a temporally static or varying, inhomogeneous, magneticand/or inhomogeneous electric field in the sample, so that thefluorescent marker comes across locally different conditions.

Excitation of fluorescence radiation in the sample, for example byprimary radiation of suitable quantum energy.

Generation of at least one optical image of the fluorescence radiationcoming from the sample.

Calculation of the spatial distribution of the fluorescent marker bymeans of the at least one above-mentioned image and by means of theknown associated strength distribution of the inhomogeneous field.Preferably, the calculation is based on at least two images in the caseof spatially different field-strength distributions. If thefield-strength distributions are such that they each allow analysis of apoint-form region or pixel/voxel, normally N different field-strengthdistributions are required for the representation of N pixels/voxels.

The method concerns in a general way the steps that can be performedwith a microscope arrangement of the kind described above. With regardto details, advantages and further aspects of the method, reference ismade in particular to the above description. The method enablesinformation to be extracted from the sample with high local resolution,by locally varying the conditions of the fluorescence within the sampleby means of a spatially inhomogeneous field and the use of a fluorescentmarker sensitive thereto.

If an inhomogeneous magnetic field is used in the method, it preferablyhas (at one point at least) a gradient of at least 1 ² T/m, especiallypreferably of at least 10³ T/m, and very especially preferably of atleast 10⁶ T/m. At such values of the gradient, the magnetic fieldstrength per nanometer varies by about 0.1 to 1 mT, wherein knownmagnetically sensitive fluorescent markers already respond to suchchanges. At said gradients, a spatial resolution in the region of 1 nmcan be achieved.

If an inhomogeneous electric field is used in the method, thispreferably has (at one point at least) a gradient of at least 10¹¹ V/m²,especially preferably of at least 10¹⁵ V/m². At these values and withthe use of ordinary electrically sensitive fluorescent markers, aresolution in the nanometer range is likewise obtained.

According to a preferred embodiment of the method, the inhomogeneousfield is configured so that it has a local minimum of the fieldstrength. In particular, this minimum can have the value zero, that is,can correspond to a field-free region. Preferably, the width of thelocal minimum is smaller than the optical resolution of the fluorescencemicroscope. Here, the “width” of the minimum is expediently defined independence on the effect of the field on the fluorescent marker underconsideration. If the latter, for example, in the case of a vanishingfield has a minimal fluorescence yield that increases to a maximum valueas the field increases, the “width” can be defined as the region havinga fluorescence yield below a specific percentage (for example, 50%) ofthe maximum value. In the spatially confined region of the localminimum, special conditions are therefore created for the fluorescence,which lead to observable effects in the fluorescence radiation emittedfrom the sample. The minimum can therefore be used as the focal regionfor targeted analysis of small volumes within the sample.

According to another aspect of the method, the sample is located duringits analysis in a solution that contains the fluorescent marker. In thisway, fluorescent marker that has been lost and/or degraded, for example,by bleaching, is constantly replaced from the solution, so that thefluorescence in the sample can be maintained for a comparatively longperiod.

These and other aspects of the invention are apparent from and will beelucidated with reference to the embodiments described hereinafter.

In the drawings, the single FIGURE shows schematically the principle ofthe microscope arrangement according to the invention and its use.

Within the field of fluorescence microscopy, the point is to determinethe distribution of a fluorescent marker 21 within a sample 20; thisdistribution, for example, in the case of biological samples, canprovide information about anatomical and/or metabolic conditions.Suitable fluorescence microscopes are found, for example, in the LSM 510series of the firm Carl Zeiss in Oberkochen. To excite the fluorescence,the sample 20 is irradiated with (primary) photons ν_(E), which areabsorbed by the atoms and molecules of the fluorescent marker 21 andthus send them into an excited energy state. This energy state is thendegraded again through emission of the fluorescence photons ν_(F), whichhave a wavelength characteristic of the marker.

From the fluorescence photons ν_(F) entering an associated microscope10, an image of the intensity distribution of the fluorescence radiationis created by the optical system of the microscope. On this image, anobserver can detect by eye, for example, areas of increasedconcentration of the fluorescent marker. Moreover, in the case ofadvanced evaluation methods as a rule the intensity distribution of thefluorescence radiation is measured quantitatively. Common to all knownfluorescence microscopes operating according to the principle describedso far is the fact that the optical resolution is limited to values ofabout 100 to 30 nm.

To overcome this limitation, with the arrangement illustrated in theFIGURE it is proposed to use a magnetically sensitive fluorescent marker21 as well as a spatially inhomogeneous magnetic field 33 within thesample 20. Magnetically sensitive fluorescent markers alter theirfluorescence behavior in dependence on the strength of the externalmagnetic field in which they are located. Typical examples of suchfluorescent markers are the so-called “exciplexes”, which are formedfrom excited complexes. That is to say, a molecule crosses through aprimary photon into an excited state and combines with another moleculeto form a dimer. In the dimer, the energy levels of the singulet andtriplet states are virtually degenerate and shuffle in the course oftime. If an external magnetic field is used, the triplet state splitsinto three different states, whereby the speed of mixing of singulet andtriplet states is changed. The exciplex can cross into the ground statewhile emitting a fluorescence photon, the probability that this photonwill be emitted being dependent on whether a singulet or triplet statewas present. In this way, the fluorescence yield also depends on theexternal magnetic field. Changing of the fluorescence by the magneticfield can here amount to more than 30%, and effects can be achievedalready with fields of less than 2 mT. Ideally, the two reactionpartners of the exciplex are chemically bonded with one another to forma so-called intramolecular exciplex. Examples of magnetically sensitivefluorescent markers are known also from studies relating to MARY(MAgnetic field effect on Reaction Yield) spectroscopy, cf. GünterGrampp et al.: RIEKEN Review No. 44 (February 2002)). Further, one canrefer in this regard to the publications of N. Kh. Petrov (e.g.: N. Kh.Petrov, V. N. Borisenko, A. V. Starostin, M. V. Alfimov, Amplificationof the cage effect in binary solvents detected by technique of magneticmodulation of exciplex fluorescence, Izv. AN SSSR, ser. chim., 1991, no.11, p. 2456;

N. Kh. Petrov, V. N. Borisenko, A. V. Starostin, M. V. Alfimov, Polarmolecular clusters produced upon photoinduced electron transfer in anintermolecular exciplex in binary solvents, J. Phys. Chem., 1992, vol.96, no. 7, p. 2901;

N. Kh. Petrov, V. N. Borisenko, M. V. Alfimov, Study of preferentialsolvation in binary solvent mixtures by the fluorescence-detectedmagnetic field effect. J. Chem. Soc., Faraday Trans., 1994, vol. 90, no.1, 109-111; N. Kh. Petrov, V. N. Borisenko, M. V. Alfimov, MagneticField Effects of Exciplex Fluorescence of the Pyrene-Azacrown EtherSystem in the Presence of Alkali and Alkaline Earth Salts, J. Chem. Soc.Mendeleev Commun. 1995; N. Kh. Petrov, V. N. Borisenko, M. V. Alfimov,T. Fiebig, H. Staerk, Fluorescence-detected Magnetic Field Effects inExciplex Systems Containing Azacrown Ethers as Electron Donor, J. Phys.chem., 1996, vol. 100, no. 16, 6368-6370.

Alternatively, instead of magnetically sensitive fluorescent markers,electrically sensitive fluorescent markers could be used. These arecorrespondingly distinguished by the fact that their fluorescencebehavior depends on the external electric field in which they arelocated. Demonstrable changes in the fluorescence behavior frequentlyoccur already at differences in the electric field strength of the orderof magnitude of 10⁶ V/m. Electrically sensitive fluorescent markers arealso used for measuring naturally occurring electric field strengths(for example, within a cell membrane). For that purpose, electricallysensitive fluorescent markers are described, for example, in UnitedStates patent specification 2002/0155520 A9; the markers describedtherein can be used also in the present case and the document is fullyincorporated by reference into the present application. Furthermore, asregards this subject matter reference can be made to Jian-young Wu etal., Histochemical Journal, 30 169-187 (1998) as representative offurther publications. For the record, the method described below formagnetically sensitive fluorescent markers can be carried outanalogously for electrically sensitive markers as well.

To be able to exploit the sensitivity of the fluorescent marker 21 tomagnetic fields, a field generator 30 that is capable of generating aninhomogeneous magnetic field 33 within the sample 20 is positioned inthe vicinity of the probe 20. In the example illustrated, the fieldgenerator 30 consists of three (for example, permanently magnetic) polebodies. A first pole body 31 having the polarity “magnetic North”preferably has a tip in order to improve the optical accessibility tothe sample. On opposite sides of the first pole body 31 there are twofurther pole bodies 32 having the polarity “magnetic South”. These couldalternatively annularly surround the first pole body 31. With thisconfiguration, a focal region 22, in which the magnetic field strengthis approximately zero, occurs in front of the tip of the first pole body31, as indicated by dashed field lines 33. The distance of the tip fromthe focal region 22 depends on the desired gradient and for gradients of10⁶ T/m is typically about 1 μm and, for gradients of 10 T/m, in themillimeter range. The width of the focal region can be, for example,about 1 nm. Furthermore, the field generator 30 is dimensioned so thatthe gradient of the magnetic field 33 around the focal region 22 is morethan 10⁶ T/m (in the case of an inhomogeneous electric field, thegradient should be more than 10¹⁵ V/m²).

In the FIGURE, above the microscope arrangement the distribution of theintensity I_(F) of the fluorescence radiation ν_(F) from the sample 20over the location x is reproduced. It is assumed in the example herethat the fluorescent marker 21 is approximately uniformly distributed inthe sample 20, so that in principle all points emit fluorescenceradiation with the same intensity. Excluded from this, however, is thesmall focal region 22 of the inhomogeneous magnetic field 33, in whichthe intensity is reduced. At the associated point x₀ the intensitydistribution I_(F) thus exhibits a minimum. But because of the limitedoptical resolution of the microscope 10, the minimum cannot be sharplyreproduced directly. Rather, the course of the fluorescence intensityI_(FM) observed with the microscope arrangement has the courseillustrated in the upper diagram of the FIGURE, in which the minimum iscorrespondingly broadened and flattened. This “blurred” reproduction ofa focal region 22 can nevertheless be used to improve the resolution ofthe microscope arrangement, since the position of the inhomogeneousmagnetic field 33 and hence of the focal region 22 within the sample canbe varied. By simultaneously observing the changes in the measuredintensity distribution I_(FM), mathematical conclusions can be drawnabout the conditions in the locally defined region 22. As a result, bymoving the focal region 22, the concentration of the fluorescent markercan be scanned with a resolution in the nanometer range.

To optimize the reproduction quality achievable with the microscopearrangement, the proportion of background radiation should preferably beminimized and hence the signal-noise ratio maximized. One way to do thiscomprises exciting only a small sample region by primary radiationν_(E), and restricting the observation to a correspondingly smallregion. Ideally, a high-quality confocal scanning microscope istherefore used. Furthermore, techniques such as two-photon excitationand a stimulated emission are helpful. The signal-noise ratio can alsobe improved by integration over time and/or a high light level for theexcitation. In this respect photobleaching of the fluorescent markerconstrains this method. Like all the above-mentioned techniques, ingeneral also the numerous other techniques known from fluorescencemicroscopy can be used in combination with the present method.

Moreover, it may be that the fluorescent marker 21 will degenerate overthe course of time as result of chemical reaction of the excited states(for example with oxygen). In that case, the degenerate marker moleculesare replaced preferably by way of diffusion. If, for example, thesurface of a sample is to be observed, this can be immersed in asolution of the fluorescent marker. The molecules of the fluorescentmarker then absorb at the surface of the sample, degenerate moleculesbeing replaced from time to time by unconsumed molecules of thefluorescent marker from the solution.

The described microscope arrangement can be used in the imaging of solidbodies, similar to the way scanning currently occurs with electronmicroscopes. It is advantageous in the above-described magneticfield-based method that the sample does not need to be dried, andbiological samples can even still be living. If an electric field isused, the sample should be electrically isolated. This can be achieved,for example, by placing it in oil or demineralized water or by freezing.

Moreover, the method can be used for detection of biological molecules.In that case, a sample of different molecules could be mixed withfluorescent markers (one or more colors), which bond specifically ornon-specifically to the molecules to be identified. Identification ofthe molecules would then be effected by means of the observed spatialdistribution of the fluorescent markers. In the case of a non-specificbond of the fluorescent marker to the molecules, large molecules couldbe identified, for example, by the fact that the fluorescent markerbonds to different points of the molecule and therefore renders thecharacteristic spatial form of the molecule recognizable. In the case ofa specific bond of fluorescent markers, the method could, for example,allow rapid segmentation of a DNA sample; here, different colors couldencode different nucleotides.

1. A microscope arrangement for imaging a sample (20) that contains amagnetically and/or electrically sensitive fluorescent marker (21),comprising a fluorescence microscope (10) for exciting and imagingfluorescence radiation (ν_(F)) from the sample (20); a field generator(30) for generating an inhomogeneous magnetic and/or inhomogeneouselectric field (33) in the sample (20).
 2. A microscope arrangement asclaimed in claim 1, which is designed to alter the inhomogeneous field(33) within the sample (20) in a defined manner.
 3. A microscopearrangement as claimed in claim 1, characterized in that the fieldgenerator (30) for generating an inhomogeneous field (33) has a firstpole body (31) of a first polarity (N), which on at least two oppositesides is adjacent to second pole bodies (32) of different polarity (S).4. A microscope arrangement as claimed in claim 1, characterized in thatit comprises a data processing device for image processing of the image(I_(FM)) recorded by the fluorescence microscope (10), the dataprocessing device being designed to reconstruct the distribution of thefluorescent marker (21) in the sample (20) from the known spatialstrength distribution of the inhomogeneous field (33) during one orpreferably several recordings.
 5. A method of determining the spatialdistribution of a magnetically and/or electrically sensitive fluorescentmarker (21) in a sample (20), which method comprises the followingsteps: generation of an inhomogeneous magnetic and/or inhomogeneouselectric field (33) in the sample (20); excitation of fluorescenceradiation (ν_(F)) in the sample (20); generation by means of afluorescence microscope (10) of an image (I_(FM)) of the fluorescenceradiation (ν_(F)) coming from the sample (20); calculation of thespatial distribution of the fluorescent marker (21) by means of thegenerated image (I_(FM)) and by means of the known strength distributionof the field (33).
 6. A method as claimed in claim 5, characterized inthat the inhomogeneous magnetic field (33) has a gradient of at least10² T/m, preferably of at least 10⁶ T/m.
 7. A method as claimed in claim5, characterized in that the inhomogeneous electric field has a gradientof at least 10¹¹ V/m², preferably of at least 10¹⁵ V/m².
 8. A method asclaimed in claim 5, characterized in that the inhomogeneous field (33)has a local minimum (22) of field strength, especially a field-freepoint or region.
 9. A method as claimed in claim 8, characterized inthat the width of the local minimum (22) is smaller than the opticalresolution of the fluorescence microscope (10).
 10. A method as claimedin claim 5, characterized in that the sample (20) is located in asolution with the fluorescent marker.